The quantitative detection of DNA strand breaks is of great significance for many fields of biomedical research and diagnosis already today. The mechanisms of DNA-damage and DNA repair and their disturbances can be investigated via the detection of DNA strand breaks. Furthermore, it is thereby possible to carry out toxicological screening of substances such as chemicals, natural substances or pharmaceutical preparations. Moreover, various cell and tissue samples from patients can be examined for DNA damage and DNA repair capacity, respectively, by quantifying DNA strand breaks. This is of interest for what is called monitoring the effectiveness of radiation therapy and cytostatic chemotherapy, respectively, in the malignant cells to be killed before and during the treatment. It is also possible to evaluate the side-effect risk of radiation therapy and chemotherapy, respectively, before the therapy is started by examining normal (nonmalignant) cells as to DNA strand breaks. The examination of DNA strand breaks is also significant within the scope of preventive medicine for the screening for individuals having a high degree of DNA damage and low DNA repair capacity, respectively. A greater cancer risk has to be assumed for these persons, so that an especially close-knit preventative program is indicated for them. In this connection, what is called biomonitoring must also be considered in industrial medicine. Here, standardized DNA damage treatments would each be made with the proband's cell material to be tested.
So far, there have been some methods of examining DNA strand breaks, which are, however, very time-consuming and labor-intensive, since they must mostly be carried out manually. Another drawback of these methods is the care which must urgently be taken in the various steps so as not to reach false-positive or false-negative results. The "alkaline elution" has to be mentioned as a known method of examining DNA strand breaks. Here, a controlled, alkaline denaturation of the DNA is induced in a cell lysate. The strand break frequency is measured by determining the elution rate of the single-stranded DNA fragments through a suitable polycarbonate filter. Another known method is what is called the "comet assay". This is an in situ assay which can be carried out in various ways. In this case, cells must be embedded in an agarose gel. Following lysis "in situ" an electric field is applied, which results in a more or less marked migration of chromatin out of the nucleus. The microscopically readable migration path is considered a standard that applies to the number of DNA strand breaks. In addition, the "Fluorescence-detected Alkaline DNA Unwinding" (Birnboim, H. C. and Jevcak, J. J., 1981, Cancer Res. 41:1889-1892, abbreviated as "FADU-Assay", is also known for measuring DNA strand breaks. Here, test cells are lysed and the cellular DNA exposed in this way, which depending on the pretreatment of the cells has a more or less large number of single-strand or double-strand breaks is then subjected to denaturation under accurately controlled conditions, whereby the DNA double strand is converted into single strands. In practice, this is effected by an extremely careful piling-up of an alkaline solution, each mixture with the lower phase (the lysate) having to be avoided by all means. Within some minutes, part of the alkaline solution diffuses into the lysate. The alkaline denaturation then starts in each case from the DNA break ends and chromosome ends, respectively, and proceeds linearly, namely in both orientations in the case of a single-strand break. After stopping this alkaline DNA-unwinding by neutralization, the rest of the non-denatured DNA which remains in the sample is measured with non-denatured DNA via the intensity of the ethidium bromide fluorescence. The amount of denatured DNA calculated therefrom is a direct standard that is applied to the number of DNA strand breaks present at the time of cell lysis. The fluorescence measurement is standardized so that it applies (i) to those cell lysates (referred to as "T samples") in which no denatured pH was reached because the neutralization buffer had been added prior to the addition of the alkaline solution and (ii) to those lysates (referred to as "B samples") which had been provided with a very large number of DNA breaks by DNA shearing (e.g., by means of ultrasound treatment) prior to the alkaline denaturation. In the case of the T sample the content of denatured DNA is set to 0%, whereas the content of denatured DNA in the B sample is set to 100%. However, the FADU assay is very labor-intensive and susceptible to failure because several high-precision pipetting steps and the accurate observance of time and temperature conditions are required for each individual sample. In some steps attention has to be paid to the fact that the contents of the sample tubes comprising several piled-up liquid phases are not mixed. With regard to the required quadruple parallel determinations, the T and B samples which always have to be carried along and the large number of samples to be determined this adds up to an immense pipetting work, each pipetting step having to be carried out with the utmost constant care. In this connection, it is aggravating that the FADU assay must be carried out while the laboratory is darkened, since the cell lysates are very sensitive to light.
As explained already, the drawback of all of these methods is that they are very labor-intensive and the sample throughput per time unit is not very high. Thus, about 3 manhours are required to manually carry out the FADU assay with 30 samples. As regards the alkaline elution 16 hours 2 of them manhours) have to be estimated for about 36 samples.
In the case of the comet assay even 8 manhours are required to process only 20 samples.
Therefore, it is the object of the present invention to provide a method of quantifying DNA strand breaks, which can be carried out easily, has a good sample throughput per time unit and supplies reliable results. The object of the present invention also consists in providing a device by means of which the method can be carried out.